The localization of FGFR3 mutations causing thanatophoric dysplasia type I differentially affects phosphorylation, processing and ubiquitylation of the receptor

Abstract

Fibroblast growth factor receptor 3 (FGFR3) belongs to a family of four genes (FGFR1-4) encoding receptors with tyrosine kinase activity (RTK). These structurally related proteins exhibit an extracellular domain (ECD) composed of three immunoglobin-like domains, an acid box, a single transmembrane domain and a Recurrent missense fibroblast growth factor receptor 3 (FGFR3) mutations have been ascribed to skeletal dysplasias of variable severity including the lethal neonatal thanatophoric dysplasia types I (TDI) and II (TDII). To elucidate the role of activating mutations causing TDI on receptor trafficking and endocytosis, a series of four mutants located in different domains of the receptor were generated and transiently expressed. The putatively elongated X807R receptor was identified as three isoforms. The fully glycosylated mature isoform was constitutively but mildly phosphorylated. Similarly, mutations affecting the extracellular domain (R248C and Y373C) induced moderate constitutive receptor phosphorylation. By contrast, the K650M mutation affecting the tyrosine kinase 2 (TK2) domain produced heavy phosphorylation of the nonglycosylated and mannose-rich isoforms that impaired receptor trafficking through the Golgi network. This resulted in defective expression of the mature isoform at the cell surface. Normal processing was rescued by tyrosine kinase inhibitor treatment. Internalization of the R248C and Y373C mutant receptors, which form stable disulfide-bonded dimers at the cell surface was less efficient than the wild-type, whereas ubiquitylation was markedly increased but apparently independent of the E3 ubiquitin-ligase casitas B-lineage lymphoma (c-Cbl). Constitutive phosphorylation of c-Cbl by the K650M mutant appeared to be related to the intracellular retention of the receptor. Therefore, although mutation K650M affecting the TK2 domain induces defective targeting of the overphosphorylated receptor, a different mechanism characterized by receptor retention at the plasma membrane, excessive ubiquitylation and reduced degradation results from mutations that affect the extracellular domain and the stop codon.

The FGFR Landscape in Cancer: Analysis of 4,853 Tumors by Next-Generation Sequencing

“…For both of these mutations, the creation of a new, unpaired cysteine residue results in formation of interreceptor disulfide bones, increased homodimerization and signaling (34). In vitro, FGFR3 R248C promotes increased cell numbers at confluence, induces proliferation, induces morphologic transformation, reduces apoptosis, and decreases attachment to fibronectin, but does not alter migration (34,35). FGFR3 G370C lies in the extracellular juxtamembrane region.…”

“…We are unaware of data regarding the ability of this mutation to transform cells in vitro. FGFR K650E also shows ligand independent activation, although by undefined mechanism(s) (35,36) and is able to transform NIH3T3 cells (37,38). FGFR3 Y373C is also thought to induce disulfide bond formation causing constitutive activation of the receptor (32,35).…”

Receptor tyrosine kinase transmembrane domains

“…A more recent study addressed FGFR3 TM mutations to cysteine residues, Tyr373Cys, Ser371Cys and Gly370Cys, all linked to thanatophoric dysplasia I (TD I). 33 These residues are most likely in the bilayer headgroup region of the membrane, and are sometimes assigned as part of FGFR3 extracellular domain. The mutations cause a downregulation defect, which is currently believed to be the major determinant of the pathology.…”

“…The mutations cause a downregulation defect, which is currently believed to be the major determinant of the pathology. 33 In addition, mutations to Cys, such as the TD mutations described above and the rare Gly375Cys FGFR3…”

Unfoldomics of Human Genetic Diseases: Illustrative Examples of Ordered and Intrinsically Disordered Members of the Human Diseasome

“…Two forms of congenital dwarfism, Chondrodysplasia and Hypochondroplasia, as well as such skeletal developmental pathologies as Thanatophoric dysplasia (a severe inhered disease characterized by extremely short limbs and folds of extra skin on the arms and legs), and Craniosynostosis (a defect in which one or more of the flexible and fibrous joints between the skull bones closes too soon, which stops the normal capacity of the skull to expand in early childhood) are due to the genetic defects in the fibroblast growth factor receptor 3 gene (FGFR3) [58]. This involvement of a FGFR3 gene in a number of skeletal diseases is definitely due to the hub-like functionality of its product.…”